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Revista Brasileira de Anestesiologia

Print version ISSN 0034-7094On-line version ISSN 1806-907X

Rev. Bras. Anestesiol. vol.56 no.5 Campinas Sept./Oct. 2006 



Complexation of 50% enantiomeric excess (S75-R25) bupivacaine with cyclodextrins and spinal block anesthesia in rats*


Mezcla con exceso enantiomérico de 50% (S75-R25) de bupivacaina con complejo de ciclodextrinas y anestesia por vía subaracnoidea en ratones



Daniele Ribeiro de Araujo, M.D.I; Angélica de Fátima de Assunção Braga TSA, M.D.II; Carolina Morales Moraes, M.D.III; Leonardo Fernandes Fraceto, M.D.IV; Eneida de Paula, M.D.V

IDoutora em Biologia Funcional e Molecular Bioquímica, Instituto de Biologia-UNICAMP
IIProfessora-Associada do Departamento de Anestesiologia, FCM-UNICAMP
IIIGraduanda do Curso de Farmácia, Universidade de Sorocaba – UNISO
IVProfessor Doutor do Curso de Farmácia, Universidade de Sorocaba – UNISO
VProfessora-Associada do Departamento de Bioquímica – Instituto de Biologia, UNICAMP

Correspondence to




BACKGROUND AND OBJECTIVES: In order to prolong the action and reduce systemic toxicity, formulations of local anesthetic (LA) complexed with cyclodextrins (CD) have been developed. This study determined the physical-chemical characterization and evaluated the effects of inclusion complexes of racemic bupivacaine (S50-R50) and 50% enantiomeric excess (S75-R25) bupivacaine with hydroxypropil-beta-cyclodextrin (HP-b-CD) in rats, and comparing them with the solutions currently used in the clinical practice.
METHODS: Inclusion complexation of S75-R25 with HP-b-CD (equimolar ratio 1:1) was characterized by phase-solubility studies varying the concentrations of HP-b-CD and the temperature. Affinity constants (K) for HP-b-CD and the thermodynamic parameters for complexation were determined. Motor and sensitive anesthesias were evaluated through the subarachnoid administration of the formulations in the concentration of 0.5%.
RESULTS: Inclusion complexation was observed through the increase in aqueous solubility of LA in different temperatures and concentrations of HP-b-CD. The in vivo tests demonstrated that S50-R50HP-b-CD and S75-R25HP-b-CD reduced latency (p < 0.001) without changing the recovery time of the motor block, time for maximal effect, and total effect of the drugs. Besides, both formulations increased the intensity (1.5 times, p < 0.001) and prolonged the duration of analgesia compared to the free drugs.
CONCLUSIONS: The complexes S50-R50HP-b-CD and S75-R25HP-b-CD potentiated the differential nervous block, and can be used to reduce the frequency of administration or the dose of the LA to induce the same effect. The formulation containing enantiomeric excess (S75-R25) bupivacaine showed to be interesting in the development of safer formulations, and useful for the treatment of acute pain in the postoperative period.

Key Words: ANESTHETICS, Local: bupivacaine; ANIMALS: rats.


JUSTIFICATIVA Y OBJETIVOS: Con la finalidad de prolongar la duración de la acción y reducir la toxicidad sistémica, se han desarrollado formulaciones de AL con complejo de ciclodextrinas (CD). Ese estudio realizó la caracterización físico-química y evaluó en ratones, los efectos de los complejos de inclusión de bupivacaína racémica (S50-R50) y de la mezcla con exceso enantiomérico de 50% (S75-R25) de bupivacaína con hidroxipropil-betaciclodextrina (HP-b-CD), comparándolos con las soluciones actualmente utilizadas en la clínica.
MÉTODO: Los complejos de inclusión de S75-R25 en HP-b-CD (razón molar 1:1) fueron caracterizados por estudios de solubilidad de fases variando las concentraciones de HP-b-CD y la temperatura. Fueron determinadas las constantes de afinidad (K) por la HP-b-CD y los parámetros termodinámicos para los complejos. Los bloqueos motor y sensitivo fueron evaluados en ratones, a través de la administración subaracnoidea de las formulaciones en la concentración clínica de 0,5%.
RESULTADOS: La formación de complejos de inclusión se observó a través del aumento de la solubilidad acuosa del AL bajo diferentes temperaturas y concentraciones de HP-b-CD. Las pruebas in vivo mostraron que S50-R50HP-b-CD y S75-R25HP-b-CD redujeron la latencia (p < 0,001) sin alterar el tiempo de recuperación del bloqueo motor, tiempo para efecto máximo y efecto total de los fármacos. Además, ambas formulaciones aumentaron la intensidad (1,5 veces, p < 0,001) y prolongaron la duración de la analgesia, con relación a los fármacos libres.
CONCLUSIONES: Los complejos, S50-R50HP-b-CD y S75-R25HP-b-CD, potenciaron el bloqueo nervioso diferencial, pudiendo ser utilizados para reducir la frecuencia de administraciones o la dosis de AL para inducción de un mismo efecto. La formulación conteniendo la mezcla con exceso enantiomérico de 50% (S75-R25) de bupivacaina fue interesante en el desarrollo de formulaciones seguras y útiles para el tratamiento del dolor agudo en el período postoperatorio.




The advances in basic and clinical research have expanded the pharmacological options for pain treatment and local anesthetics (LA), which have the ability to block the excitation-conduction process in peripheral nerves, are among the several classes of drugs used to alleviate or eliminate pain. Even though bupivacaine has a great clinical applicability, ropivacaine, levobupivacaine, and, more recently, the 50% enantiomeric excess (S75-R25) bupivacaine have become new options for prolonged regional anesthesia. However, the LAs currently used present limitations due to their relatively short action and their toxicity for the central nervous and cardiovascular systems 1,2.

Aiming at prolonging the duration of action and reducing systemic toxicity of these drugs, several formulations of LA complexed with cyclodextrins (CD), cyclic oligosaccharides resulting from the enzymatic hydrolysis of starch 3,4, have been developed. Besides the physical-chemical stabilization, the main effects of complexation with CD are observed especially in the change in aqueous solubility, the liberation ratio, and pharmacokinetics and pharmacodynamic properties, modifying the duration and intensity of the pharmacological effects of these drugs 5,6.

Animal studies indicate that complexation with beta-cyclodextrins (b-CD) and some of its derivatives, such as hydroxypropil-beta-cyclodextrin (HP-b-CD) (Figure 1), increases aqueous solubility and the duration of the nervous block induced by racemic bupivacaine 7-11. Furthermore, a recent study demonstrated that complexation of racemic bupivacaine (S50-R50) and the 50% enantiomeric excess (S75-R25) with HP-b-CD potentiated the intensity and duration of analgesia after sciatic nerve block in mice, being potentially advantageous for the relief of postoperative pain 12.

Since bupivacaine (S75-R25) is less toxic that racemic bupivacaine (S50-R50), the objective of this study was to determine the physical-chemical characteristics and the effects of the spinal block administration in rats of the inclusion complexation of racemic bupivacaine (S50-R50) and enantiomeric mixture (S75-R25) with HP-b-CD, and comparing them with the free solutions currently used.



The solid complexes were prepared mixing proper doses of HP-b-CD and S75-R25 in water in a molar ratio of 1:1. Samples were agitated for 24 hours at room temperature. Afterwards, the solution was lyophilized and stored at 20° C for later use. Enough doses of the solid complexes were weighed to obtain the proper concentrations and dissolved, afterwards, in a buffer (20 mM of HEPES with pH of 7.4 with 154 mM of NaCl). The physical-chemical complex was characterized through solubility studies according to the method described in the literature 13. Excess local anesthetic (15 mM) was added to the buffer solution, 50 mM with pH of 7.4, in the presence of increasing doses of HP-b-CD (0, 5, 10, 20, 25, 30, 35 nM).

Solutions were agitated at 110 rpm at different temperatures (10°, 15°, 20°, 30°, and 40° C) until the system reached an equilibrium (24 h), and the concentration of the anesthetic dissolved was determined by spectrophotometry (molar absorbance, e = 470) 14. With these data, we obtained the solubility isotherms, and the affinity constants (K) were measured at different temperatures using equation 1, where: So is the initial concentration of the LA in solution.

A van't Hoff chart was built using the affinity constant (K) against the temperature and measuring the thermodynamic parameters for the complex in question: enthalpy (DH°) and entropy (DS°), according to equation 2, where T is the temperature (in degrees Kelvin) and R is the ideal gas constant (8.31 kJ/mol) 15-17.

This experimental study was conducted after approval by the Ethics Committee on Animal Experiments of the Instituto de Biologia of the Universidade Estadual de Campinas (CEEA) – IB – UNICAMP, which follows the rules of the Colégio Brasileiro de Experimentação Animal (COBEA). Groups of five male, albino rats, of the Wistar lineage, approximately 90 days old, weight varying from 200 to 250 g, were used. The animals came from the Centro de Bioterismo of UNICAMP (CEMIB) and were exposed to light/dark, 12-hour cycles, with free access to food and water, housed in groups (5 animals per cage), and acclimated to the local of the experiment for at least 7 days.

The animals were firmly contained while a needle connected to a Hamilton syringe was introduced perpendicularly in the L5-L6 space (Figure 2) to perform the subarachnoid injection. The injection site was restricted to the region where the spinal cord ends and the cauda equina begins, to reduce the possibility of spinal lesion and to facilitate intervertebral access. When the needle was inserted in the subarachnoid space, there was a sudden movement of the tail. This reflex was considered an indication that the puncture was successful. A maximun of 20 µL of the formulations was injected 18.



Motor and sensitive blocks were evaluated simultaneously and every measurement was done by the same investigator. Animals were observed for 24 h after the treatment to determine the incidence of systemic (seizures and deaths) or local (failure to recover normal movements) toxic effects. Experimental groups were characterized as follows:

  • Control Group: HP-b-CD;
  • S50-R50 Group: racemic bupivacaine;
  • S75-R25 Group: 50% enantiomeric excess bupivacaine;
  • S50-R50HP-b-CD (1:1) Group: racemic bupivacaine with hydroxipropil-b-cyclodextrine at a 1:1 molar ratio (S50-R50:HP-b-CD);
  • S75-R25HP-b-CD (1:1) Group: 50% enantiomeric excess bupivacaine with hydroxipropil-b-cyclodextrine at a 1:1 molar ratio (S75-R25:HP-b-CD).

The presence of motor block was determined by "dragging" the hind legs and/or "closing" of the fingers 19, and its intensity was evaluated according to the following scores: 0 (normal use of the hind legs), 1 (unable to flexion completely the hind legs), and 2 (cannot use the hind legs). Animals were evaluated every minute for the first 5 minutes after the subarachnoid administration, followed by evaluation at 10-minute intervals until complete recovery of the movements (observation for at least 1 hour). Parameters evaluated were latency (interval between the injection and development of motor block grade 1 or 2), length of time to reach maximum score (Tmax), length of time to recover motor function, and total effect of the LA (estimated by the area under the effect-time curve, AUC).

Sensitive block was evaluated by a mechanical stimulus 20. A device called analgesimeter, which generates a gradual increase in the strength (in grams) used by a plastic extremity over the dorsal surface of the animal's paw, was used. To avoid the analgesia produced by stress, each animal was wrapped in a towel, except for its head and legs. Paw withdrawal reflex was considered an indication of the pain threshold – Pain Withdrawal Threshold to Pressure (PWTP). Basal PWTP was measured before the experiment and animals with a pain threshold below or above 90 to 140 g were excluded, and a maximal pressure (cut-off) of 350 g, considered as representative of a sensitive block, was established. After the administration of the excipient or formulations, measurements were done for five hours; at 15-minute intervals for the 1st hour, 30-minute intervals for the 2nd and 3rd hours, and 60-minute intervals in the last two hours. Analgesia was defined as an increase in the pressure threshold of the animals at least 50% greater than in the group treated with the excipient. The end-point of analgesia was established when there were no statistical differences among the test and control groups 12.

The Mann-Whitney test was used for the statistical analysis of the motor block (latency, Tmax, recovery time, and AUC) and the data expressed as median (minimal and maximal limits). PWTP values during sensitive blocks were expressed as mean ± SD and the individual times were evaluated by the Variance Analysis (One-way ANOVA) and posteriorly by the Tukey-Kramer test 21. A value was considered statistically significant when p < 0.05.



It was possible to observe, through the solubility phase tests, the complexation of S75-R25 with HP-b-CD at a pH of 7.4 in different temperature (Figure 3). The solubility diagram of the phase systems indicates formation of soluble complexes with a linear increase in solubility of S75-R25 against the concentrations of HP-b-CD tested in different temperatures.



Using linear regressions obtained with the curves in figure 3 and equation 1, the affinity constant (K) between S75-R25 and HP-b-CD in different temperatures was calculated (Table I). The data obtained demonstrated that elevating the temperature increases the values of the affinity constant (K) due to an increase in the solubility of the drug, dislocating the equilibrium to form the complex.



A van't Hoff curve (Figure 4) was obtained from the temperature and affinity constant (K) values using equation 2, obtaining, therefore, the thermodynamic parameters of the complexation of S75-R25 with HP-b-CD.



Thermodynamic parameters were obtained from figure 4, through linear regression of the data. Values obtained for the complexation of S75-R25 with HP-b-CD were: 21.86 kJ/mol of enthalpy (DH°), 90.40 J/mol/K of entropy (DS°), and 5.07 kJ/mol of free energy in the system (DS°). The analysis of this data demonstrates that complexation between S75-R25 and the internal cavity of CD is favorable, since the free energy of the system is negative, and the entropy of the system is decisive for complexation of the drug with the CD.

Evaluation of the Motor Block

When the anesthetic solutions, free and in complexation, are administered by the subarachnoid route, they had the same effect on the animal's motor function. However, the administration of the excipient, HP-b-CD, did not produce any signs of motor block.

Comparing the experimental groups individually, we observed that the latency of the motor block was significantly reduced (p < 0.001) by the treatment with S50-R50HP-b-CD and S75-R25HP-b-CD when compared with the free solution. However, other parameters, such as length of recovery from the motor block, length of time for maximum effect, and total effect of the drug did not change with complexation with HP-b-CD (Table II).



Evaluation of the Sensitive Block

The evaluation of the sensitive block, through the PWTP, showed the effect-time relationships for the free and complexed LA. The effects of the excipient (HP-b-CD), free drugs (S50-R50 and S75-R25), and inclusion complexation (S50-R50HP-b-CD and S75-R25HP-b-CD) administered by the subarachnoid route, were evaluated (Figure 5 and 6).





The treatment with S50-R50HP-b-CD increased the nociceptive threshold when compared to the free drug, at all times of the experiment. In the initial phase (up to 180 minutes), i.e., the end point of the analgesia induced by free S50-R50, there was a significant increase (p < 0.001) in the intensity of the analgesia (approximately 1.5 times more than the free S50-R50) (Figure 5).


Table III


The inclusion complex, S75-R25HP-b-CD, also changed the answer of the animals to nociceptive stimuli, increased pain threshold and the duration of the antinociceptive action up to 5 hours after its administration (Figure 6). The intensity of the analgesia increased (1.4 times) when compared with the free S75-R25 after 180 minutes of treatment (p < 0.001).

Both systems of controlled release (S50-R50HP-b-CD and S75-R25HP-b-CD) increased the intensity and prolonged the duration of the analgesia, and the effects of the complexes was practically stable during the last two hours (Figures 5 and 6), demonstrating they are more efficient than the free S50-R50 and S75-R25.



The ability of the CDs to form inclusion complexes depends, essentially, on the compatibility of the esters and the polarity between the CD and complexed molecule. Moreover, the forces that drive the complexation have been attributed to the high energy of the repulsion forces of the water present in the cavity of the CDs, the van der Waals interactions, hydrogen bonds, and hydrophobic interactions 22,23. Complexed molecules remain oriented in the direction of the greatest contact between its hydrophobic portion and the nonpolar cavity of the CD, while its hydrophilic portion is in contact with the hydroxyl groups of the CD or exposed to the aqueous environment 3,23. In fact, the results of the physical-chemical characterization tests indicate that S75-R25 interacts with the cavity in the molecule of HP-b-CD, and that this interaction increases the aqueous solubility of the LA, being energetically favorable, and indicating a possible increase in the bioavailability of the drug.

For comparison, the groups treated with S50-R50 and S50-R50HP-b-CD were included in the tests that evaluated the anesthetic efficacy. Although complexed formulations did not change the duration or the intensity of the motor block, there was a reduction in the latency time, and a potentiating effect on the differential nervous block induced by the LA. These results agree with those obtained previously with the sciatic nervous block in mice, indicating that complexation with HP-b-CD reduces the length of time to establish motor anesthesia, promoting a faster beginning of action without prolonging or intensifying its duration 12.

The ideal LA should be long acting, have low toxicity, and greater selectivity for sensitive than motor fibers, since this characteristic of maintaining analgesia without affecting excessively motor function has great clinical relevance 24.

Evaluation of the sensitive block induced by the subarachnoid injection of inclusion complexes, S50-R50HP-b-CD and S75-R25HP- b-CD, demonstrates an increase in the intensity and duration of the analgesia when compared to the free drugs. The potentiation of the analgesic effect promoted by the complexation with CD may be connected to the increase in the total concentration of the LA available with these formulations, as well as by the formation of a barrier that hinders their binding to liquor proteins and being captured by the blood, since the free drug has a large capacity of binding to liquor proteins, therefore reducing the amount of the drug that is available.

When lipophilic drugs (such as S50-R50 and S75-R25) are administered by the subarachnoid route, they are quickly distributed in the cerebrospinal fluid. Consequently, their onset of action is faster, but the duration of their effect is shorter 25. Thus, if complexation with CD makes a greater fraction of the LA available while, at the same time, increasing the length of time it is in contact with the nerve, complexed drugs would be of great clinical utility for potentiating is analgesic action.

Even though both complexes, S50-R50HP-b-CD and S75-R25HP-b-CD, enhanced the differential nervous block, the formulation with 50% enantiomeric excess (S75-R25) bupivacaine is better than racemic bupivacaine (S50-R50) due to its lower toxic potential, favoring the development of safer formulations that are more clinically useful for the treatment of postoperative acute pain, while being administered in smaller doses.

The results of this study demonstrated that it is possible, through nano-structure formulations especially designed for each LA, to increase the differential nervous block and the total duration of the anesthesia after the administration of racemic bupivacaine (S50-R50) and 50% enantiomeric excess (S75-R25) bupivacaine. Thus, the proposed non-emulsion formulations of LAs can be used to decrease the frequency of the administration or the dose necessary to induce the same effect, which is interesting for long acting drugs with high systemic toxicity, such as bupivacaine.



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Correspondence to:
Dra. Daniele Ribeiro de Araújo
Depto. de Bioquímica Instituto de Biologia
Cidade Universitária Zeferino Vaz s/n
13083-970 Campinas, SP

Submitted for publication 30 de novembro de 2005
Accepted for publication 30 de junho de 2006



* Received from Laboratório de Biomembranas e Sistemas Biomiméticos, Departamento de Bioquímica – Instituto de Biologia, Universidade Estadual de Campinas – UNICAMP e Laboratório de Química da Universidade de Sorocaba – UNISO

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